Production of edible protein substances

ABSTRACT

The invention relates to Fusarium graminearum Schwabe deposited with the Commonwealth Mycological Institute and assigned the number IMI 145425 and variants and mutants thereof, as well as a culture medium containing the same.

This is a continuation of application Ser. No. 711,964 filed Aug. 5,1976, U.S. Pat. No. 4,061,781, which in turn is a continuation ofapplication Ser. No. 414,102 filed Nov. 8, 1973, now abandoned, which inturn is a continuation of application Ser. No. 140,303, filed May 4,1971, now abandoned.

The present invention relates to a process for the production of edibleprotein-containing substances and has particular reference to theproduction of fungal protein by microbial action.

Our copending United Kingdom Application No. 53312/66, Ser. No. 1210356,relates to a process for the production of an edible protein-containingsubstance which comprises incubating and proliferating, under aerobicconditions, an organism which is a non-toxic strain of a microfungus ofthe class Fungi Imperfecti, in a culture medium containing essentialgrowth-promoting nutrient substances, of which carbon in the form ofassimilable carbohydrate constitutes the limiting substrate inproliferation, and separating from a assimilable carbohydrate exhaustedmedium the proliferated organism which constitutes the edibleprotein-containing substance.

It is also an object of the present invention to provide fungal myceliumwhich possesses a high net protein utilisation value on rat assays of atleast 70 based on the α-amino nitrogen. However, as indicated in one ofthe examples, it is possible to have a net protein utilization value ofat least 65.

According to the present invention there is provided a process for theproduction of an edible protein-containing substance which comprisesincubating and proliferating, under acrobid conditions, a nontoxicstrain of the genus Fusarium or a variant or mutant thereof, in aculture medium containing essential growth-promoting nutrientsubstances, of which carbon in the form of assimilable carbohydrateconstitutes, the limiting substrate in proliferation, and separating theproliferated organism comprising the edible protein-containingsubstance.

The separated proliferated organism comprising the edibleprotein-containing substance may be incorporated into a foodstuff forhuman or animal consumption.

The substrate employed in the incubation stage may be of vegetableorigin, for example starch, starch containing materials or products oftheir hydrolysis, sucrose, sucrose containing materials or hydrolysedsucrose i.e. invert sugar or mixtures thereof. Thus the substrate maycomprise hydrolysed potato, molasses, glucose, hydrolysed bean starch orcassava. Alternatively substrate of animal origin comprising whey may beemployed.

The non-toxic strain of Fusarium may be a strain of Fusariumgraminearum.

The preferred non-toxic strain is our strain of Fusarium graminearumSchwabe, 10 deposited with the Commonwealth Mycological Institute andassigned the number I.M.I 145425 and variants and mutants thereof.

The following novel variants may also be employed:

I-7 deposited with the Commonwealth Mycological Institute and assignedthe number I.M.I. 154209.

I-8 deposited with the Commonwealth Mycological Institute and assignedthe number I.M.I. 154211.

I-9 deposited with the Commonwealth Mycological Institute and assignedwith number I.M.I. 154212.

I-15 deposited with the Commonwealth Mycological Institute and assignedthe number I.M.I. 154213.

I-16 deposited with the Commonwealth Mycological Institute and assignedthe number I.M.I. 154210.

Our new strain of Fusarium graminearum Schwabe, I.M.I. 145425, isnon-pathogenic to wheat. It has the following morphologicalcharacteristics:

    ______________________________________                                                                     Czapek-Dox                                                                    (Modified)                                       Media     Potato Sucrose Agar                                                                              Agar (Oxoid)                                     ______________________________________                                                250 grams of potatoes                                                         washed and diced, placed                                                      in pressure cooker 15 lbs.                                                    square inch for 15 minutes.                                                   The decoction is then                                                         squeezed through two                                                          layers of muslin. 2% of                                                       Glucose & 2% of Agar                                                          are added to the turbid                                                       filtrate and the medium                                                       autoclaved and dispersed.                                             ______________________________________                                    

Growth conditions: 25° C. several weeks

Rate of growth: 4.0 cm. in 3 days, 3.0 cm. in 3 days

Character of growth: Floccose, spreading colonies with white aerialmycelium. Substratum of PSA greyish rose with patches of crimson toyellow. Tendency to be somewhat paler on CDA. Occasionally deep redpigment produced, particularly on ageing. After one to two weeks theserial mycelium tends to become brown and collapse. The colony thenbecomes rather slimy as sporodochia are formed the colour being pink tobrown on PSA and salmon pink on CDA. No exudate is formed and pigmentformation tends to follow the mycelium colour.

Conidia: Microconidia not produced by this organism. Macroconidiaproduced from single lateral phialides or multibranched conidiophoreswith short phialides. In older cultures the conidiophores aggregate toform sporodochia, particularly on CDA. The conidia vary from falcate tocurved fusoid dorsi-ventral, septation varying from 3 to 5, commonly 5in younger culture. Spore size varies from 25-50μ×2.5μ-4.0μ.

The foot cell is often pedicellate, particularly in the longer 5 septatespores. Swollen cells occur in the mycelium and occasionallychlamydospores occur intercalary, singly or in chains.

Following is a description by way of example of the preparation ofvariants (or isolates) of Fusarium graminearum Schwabe I.M.I. 145425 andtheir morphological characteristics.

Isolates obtained from continuous culture

Fusarium graminearum Schwabe I.M.I. 154209 to 154213 were selected frommalt extract agar plates inoculated with broth from a fermenter growingFusarium graminearum Schwabe 145245 on a glucose/salts medium at 30° C.under continuous culture conditions with carbon limitation at a dilutionrate of 0.1-0.15 hr⁻¹. The fermenter was sampled at approximately 100hour intervals to assess the population of variants and the isolatesI.M.I. 154209 to 154213 were taken from the plates prepared from the1100 hour sample. These isolates are representative of the major typesof morphological variant produced during the fermentation. Under theseconditions outlined in this example the variants begin to appear onplates from 800 hours onwards. They are continually produced from thistime onward and the population slowly changes as regards the percentageof each type. The fermenter conditions are as follows:

    ______________________________________                                               Culture medium     %                                                   ______________________________________                                        Solution 1                                                                             Glucose              3.0                                                      Ammonium sulphate    0.25                                                     Potassium di-hydrogen                                                                              0.30                                                     phosphate                                                                     Magnesium sulphate   0.025                                                    Antifoam, polypropylene                                                                            0.01                                                     glycol 2000; sterilised                                                       at pH 3.0 for 30 mins.                                                        at 15 p.s.i.g.                                                       Solution 2                                                                             MnSO.sub.4 4H.sub.2 O                                                                              0.0005                                                   FeSO.sub.4 7H.sub.2 O                                                                              0.0005                                                   ZnSO.sub.4 7H.sub.2 O                                                                              0.0005                                                   CoCl.sub.2 6H.sub.2 O                                                                              0.0001                                                   CuSO.sub.4 5H.sub.2 O                                                                              0.0001                                                   Na.sub.2 MoO.sub.4 2H.sub.2 O                                                                      0.0001                                                   Sterilised 15 mins. at 15 p.s.i.g.                                   Solution 3                                                                             Biotin               0,10 or                                                                       20 μg/l                                               Choline              0,1 or 2 mg/l                                            Methionine           0 or 600 mg/l                                            Sterilised separately by filtration                                  ______________________________________                                    

All solutions were added, as necessary, aseptically to the mediumreservoir then fed to the 8.5 liter chemostat with the following growthconditions:

Temperature 30° C., aeration 1vvm (i.e., 1 volume air/volume culturemedium/minute): agitation 800 r.p.m., single 6-bladed disc turbine,0.5D, in fully baffled fermenter. The fermenter pH was maintained at 5.0by the automatic addition of sterile ammonia. Solution 3 was changed incomposition at various times to determine μmax with the variousadditions such as biotin alone or biotin plus chlorine. Growth rate 0.1to 0.15 hr⁻¹.

The five isolates have the following morphological characteristics.

    ______________________________________                                                                      Czapek Dox                                      Media     Potato Sucrose Agar Agar (Oxoid)                                    ______________________________________                                                250 grams of potatoes                                                         washed and diced, placed                                                      in pressure cooker 15 lbs.                                                    square inch for 15 minutes.                                                   The decoction is then                                                         squeezed through two layers                                                   of muslin. 2% of Glucose                                                      & 2% of Agar are added to                                                     the turbid filtrate and                                                       the medium autoclaved and                                                     dispersed.                                                            ______________________________________                                    

Growth conditions: 25° C.

Character of growth Isolate I-7

Colony morphology similar to parent A3/5 except colony diameter issmaller, 1.5 cm in three days at 30° C. on C.D.A. The floccose serialmycelium varies in degree from colony to colony but is less than I-8.Older cultures have a brown discoloration to the mycelium. Reverse,yellow-brown to greyish-rose sectors with some pigment diffusing.

Isolate I-8

Very intense white floccose aerial mycelium. Colony diameter again lessthan product A3/5, 2.0 cm in three days at 30° C. on C.D.A. Reversesalmon pink to greyish-rose segments.

Isolate I-9

Macroscopic appearance close to I-8 but reverse lighter, salmon tohyaline in colour.

Isolate I-15

Small restricted dome shape colonies. Mycelium short, tangled and withtendency to be convoluted. Many sporodochia-like structures formedgiving pink appearance at point of inoculation of streak. Pinkcoloration at periphery. Colony diameter only 0.4 cm at 3 days at 30° C.on C.D.A.

Isolate I-16

Isolate I-15 is very unstable and continually gives rise to I-16. Thisisolate has an appearance similar to I-7 although colonies tend to beslightly greater in diameter, 2.4 cm. in 3 days and more even inappearance. I-16 is more stable than I-15.

Conidia Isolate I-7

Abundant macroconidia produced in similar fashion to parent A3/5.Sporodochia formed in older cultures. Individual macroconidia similar toparent A3/5 in shape and size, 25-50μ×3.0-5.0μ. In older cultures manysporodochia are formed. Chlamydospores are also abundant in the myceliumof this isolate, intercalary and terminal. They are globose smooth10-12μ. Occasionally a single cell of a macroconidium forms achlamydospore.

Isolate I-8

Fewer macroconidia than I-7 and those present mainly smaller andsimpler, 1 to 3 septate, 25-35μ in length. Few sporodochia formed.Chlamydospores again abundant, intercalary, terminal, single and inchains.

Isolate I-9

Very similar to I-8 except more macroconidia, almost equivalent to I-7in number.

Isolate I-15

Very few macroconidia, the sporodochia-type structures are in fact madeup of packets of chlamydospores. There are also many chlamydosporespresent in the mycelium. The macroconidia are smaller than the parent,30-35μ×4μ with only 2 septa.

Isolate I-16

Very similar to I-7 with abundant macroconidia and chlamydospores. Themacroconidia are typical, 30-45μ×4μ.

The temperature of incubation in the process of the invention is ingeneral between 25° and 34° C., preferably around 30° C.

Inoculation resulting in commencement of the process is best carried outby a pregerminated seed stage comprising for example from 2% to 10% ofinoculum, usually in the range 5% to 10%.

The pH of the substrate medium during incubation is preferably keptwithin a suitable range supporting maximum growth, for example, between3.5 to 7.

The period of growth in batch culture under the above mentionedconditions is usually found to range from 20 to 48 hours. In both batchand continuous processes aeration and agitation should be carried out toprovide a sufficient level of dissolved oxygen to overcome deficiencywhich can be a limiting growth factor.

As will be well understood by those skilled in the art sufficientquantities of essential growth nutrients such as nitrogen, sulphur,phosphorus and other trace elements are maintained in the substratemedium so that growth of the substance is limited only by thecarbohydrate available to the fungus.

In addition to the nutrients stated above the presence of one or morevitamins such for example as biotin may be desirable to maintain maximumgrowth rate.

It is also desirable to add a non-toxic anti-foaming agent to thesubstrate medium to control foaming during the fermentation.

The substance produced according to the present invention may beisolated in any suitable manner known in the art. Thus the resultingmycelium may be recovered by separation, washing, filtration and drying.In this connection, however, it has been found that if the moisturecontent of the substance is reduced below a critical level of about 50%(w/w) by filtration under pressure the subsequent drying methodsemployed are not subjected to such stringent temperature limitationswhich is an important factor in the economic processing of thesematerials. The method of drying must not cause damage to the nutritionalvalue of the mycelium and may be drying in a current of air at 75° C. orfreeze drying.

The fungal mycelium produced by the process of the present inventionshows very good water binding capacity and may be useful as a thickeningand gelling agent. Not being an isolate, it retains its vitamins as wellas other nutritionally available materials such as lipids and somecarbohydrates. Fungal mycelium has satisfactory baking characteristicswhich are of value in protein enriched breads, breakfast foods and foodsnacks. The fungal mycelium, because of its filamentous structure, canbe baked, fried or puffed and presented to many communities as a foodcomparable in appearance and acceptability with conventional foods whichthey are accustomed to eating.

Following is a description by way of example of methods of carrying theinvention into effect. Examples 1-4 are of batch culture.

EXAMPLE 1

10 Liters of the following culture medium were prepared and sterilisedas described in a stirred fermenter vessel.

    ______________________________________                                        Cane molasses to provide                                                                            6% w/v sugar                                            Ammonium sulphate     1.2%                                                    NaH.sub.2 PO.sub.4    0.25%                                                   Sterilized 30 minutes 15 psig                                                 CaCO.sub.3            0.5% w/v                                                Sterilized 3 hours    15 psig                                                 ______________________________________                                    

The medium components were added aseptically and attemperated to 30° C.An inoculum equivalent to 5-10% by volume of the culture medium andgrown either on a glucose/corn steep liquor medium or other suitablematerials in shake flasks was inoculated with a spore suspension of theorganism comprising our strain of Fusarium graminearum Schwabe I.H.I.145425, and grown for 18-24 hours at 30° C. on a rotary shaker, andadded aseptically to the fermenter.

The fermenter incubated at 30° C. was then stirred at 800 rpm with a6-bladed disc turbine (0.5D) in a full baffled vessel and 1 VVM ofsterile air passed through. After 35 hours, the grown mycelium wasremoved from the fermenter, centrifuged, washed with water and dried ina warm air band drier, air temperature 75° C.

The dried product had the following composition:

    ______________________________________                                        Total Nitrogen      8.0%                                                      Ash                 5.3%                                                      Lipid               2.7%                                                      NPUop.              52      based on Total                                                                Nitrogen                                          where NPUop. is                                                               Net Protein Utilization (operative)                                           ______________________________________                                    

EXAMPLE 2

10 Liters of the following culture medium were prepared and sterilisedas described, in a 14 liter New Brunswick, Microferm, fermenter.

    ______________________________________                                                                 Final %                                              ______________________________________                                        Solution 1                                                                             Glucose          pH 3.0   3.0                                        Solution 2                                                                             Ammonium sulphate         0.7                                        Solution 3                                                                             Potassium di-hydrogen                                                                          pH 5.0   1.0                                                 phosphate                                                            Solution 4                                                                             FeSO.sub.4 7H.sub.2 O                                                                          pH 2.5   0.001                                               MnSO.sub.4 4H.sub.2 O     0.0005                                              CuSO.sub.4 5H.sub.2 O     0.0001                                              MgSO.sub.4 7H.sub.2 O     0.025                                      Solution 5                                                                             Na.sub.2 MoO.sub.4 2H.sub.2 O                                                                           0.0001                                              CoCl.sub.2 6H.sub.2 0     0.0001                                              CaCI.sub.2 2H.sub.2 O     0.0015                                     Solution 6                                                                             NaOH                      0.1                                        ______________________________________                                    

All the above solutions were sterilised by heat for 15 minutes at 15psig. Solution 7 Vitamins and/or amino acids as described belowsterilised by filtration.

The solutions were added aseptically to the vessel.

An inoculum was grown and added as in Example 1 except that the finalconcentration in the fermenter was adjusted so as to provide 0.5 gm/1dry wt. of mycelium.

The conditions of growth were temperature 30° C.; aeration 1VVM, stirrerspeed was adjusted to maintain a level of dissolved oxygen above 25% ofthe saturation value in the culture medium, measured by a New BrunswickInc. DO probe. Sterile anti-foam, polypropylene glycol 2000, was addedas required to suppress foam and pH was maintained between 6.0-6.3 bythe addition of sterile potassium hydroxide solution.

    ______________________________________                                                           Growth rates (hr,.sup.-1)                                  ______________________________________                                        (i)   Omitting solution 7 (Minimal                                                                     very slow                                                  medium)                                                                 (ii)  Solution 7 such that the final                                                                   0.2                                                        concentration of Biotin in the                                                culture medium was 50 μg/l                                           (iii) Solution 7 such that the final                                                concentration of Biotin in the                                                                   0.25                                                       culture medium was 50 μg/l;                                                Choline chloride 50 mg/l and                                                  Methionine 300 mg/l                                                     ______________________________________                                    

EXAMPLE 3

Medium and conditions were as in Example 2, but the glucose was replacedwith maltose.

    ______________________________________                                        (i)   Solution 7 as Example 2 (ii)                                                                    0.18                                                  (ii)  Solution 7 as Example 2 (iii)                                                                   0.21                                                  ______________________________________                                    

EXAMPLE 4

100 Liters of the following culture medium were prepared and sterilisedas described in a 130 l. stainless steel fermenter.

    ______________________________________                                                          % final concentration                                       ______________________________________                                        Glucose             4.0                                                       Corn steep liquor (50% Total                                                                      0.8                                                       Solids)                                                                       Ammonium sulphate   0.2                                                       Potassium di-hydrogen phosphate                                                                   0.2                                                       MgSO.sub.4 7H.sub.2 O                                                                             0.025                                                     ZnSO.sub.4 7H.sub.2 O                                                                             0.0005                                                    FeSO.sub.4 7H.sub.2 O                                                                             0.0005                                                    MnSO.sub.4 4H.sub.2 O                                                                             0.0001                                                    ______________________________________                                    

The medium was sterilised at pH 3.0 at 15 psig for 30 minutes and oncooling to 30° C. adjusted to pH 5.0 by the sterile addition of ammonia.

Biotin sterilised by filtration to give 40 μg/l final concentration, wasadded aseptically.

The vessel was inoculated with 10 liters of culture grown in a spargedvessel, for 18 hours, at 30° C., on a medium containing: Glucose 2%;tryptone (oxoid) 0.4%; yeast extract (oxoid) 0.1%; ammonium sulphate0.15%; potassium di-hydrogen phosphate 1%; sodium hydroxide 0.1%;magnesium sulphate 0.025%; ferrous sulphate 0.001%; zinc sulphate0.001%; manganese sulphate 0.0005%; copper sulphate 0.001%; anti-foam,polypropylene glycol 2000 0.05% and sterilised for 45 minutes at 15psig, inoculated with a spore suspension of our organism. Fusariumgraminearum Schwabe I.M.I. 145425.

The conditions for growth were temperature 30° C., aeration adjusted toprovide dissolved oxygen concentrations above 10% of the saturationvalue for the culture broth. Sterile anti-foam polypropylene glycol2000, was added to suppress foaming, and the pH was maintained at 5.0 bymeans of sterile ammonia additions. Samples of the mycelium taken overthe period of growth contained, on a dry weight basis: Total Nitrogen8.0-8.6%; α-Amino nitrogen 6.4-6.6%. The initial growth rate in thiscomplex medium derived from both the batched culture medium and inoculumwas approximately 0.3 hr.⁻¹.

The following Examples 5 and 6 are of continuous culture.

EXAMPLE 5

Culture medium of the following composition was prepared:

    ______________________________________                                                                Final %                                               ______________________________________                                        Solution 1                                                                    Glucose                   3.0                                                 Ammonium sulphate         0.25                                                Potassium di-hydrogen phosphate                                                                         0.3                                                 Magnesium sulphate        0.025                                               Anti-foam, polypropylene  0.01                                                glycol 2000                                                                   Sterilized at pH 3.0 for                                                      30 minutes at 15 psig                                                         Solution 2                                                                    MnSO.sub.4 4H.sub.2 O     0.0005                                              FeSO.sub.4 7H.sub.2 O     0.0005                                              ZnSO.sub.4 7H.sub.2 O     0.0005                                              CoCl.sub.2 6H.sub.2 O     0.0001                                              CuSO.sub.4 5H.sub.2 O     0.0001                                              Na.sub.2 NoO.sub.4 2H.sub.2 O                                                                           0.0001                                              Sterilized 15 minutes at 15 psig                                              Solution 3                                                                    Vitamins and/or amino acid as described below                                 sterilized by filtration.                                                     ______________________________________                                    

All solutions were added as necessary, aseptically. In 8.5 literchemostat the conditions of growth were as follows:

Temperature 30° C.; aeration 1VVM; agitation 800 rpm single 6-bladeddisc turbine 0.5D in fully baffled vessel. Organism, our strain ofFusarium graminearum Schwabe I.M.I. 145425. The pH maintained at 5.0 byautomatic addition of sterile ammonia.

    ______________________________________                                                  μ         Mycelium  NPU   NPU                                              Max. Yield   TN     AN   based based                                          hr..sup.-1                                                                         factor  %      %    on TN on AN                                ______________________________________                                        (i) Solution 3 such                                                                           0.17-  0.5   7.2  6.3  54    65                                   that the final                                                                            0.19         to   to                                              concentrate of           7.9  6.8                                             Biotin in the                                                                 culture medium                                                                was 20 μg/l                                                            (ii)                                                                              Solution 3 such                                                                           0.20-        7.7  6.1  59    78                                   that the final                                                                            0.21   0.5   to   to                                              concentration of         8.6  6.5                                             Biotin in the                                                                 culture medium                                                                was 20 μg/l and                                                            of methionine                                                                 was 600 mg/l                                                              ______________________________________                                    

EXAMPLE 6

Culture medium of the following composition was prepared:

    ______________________________________                                                              %                                                       ______________________________________                                        Bean starch (α-amylase treated)                                                                 3.0 carbohydrate                                      Corn steep liquor       1.33                                                  Ammonium sulphate       0.25                                                  Potassium di-hydrogen phosphate                                                                       0.15                                                  Magnesium sulphate      0.025                                                 Antifoam polypropylene glycol                                                                         0.025                                                 2000 (v/w)                                                                    Sterilized pH 4.0 for 30 minutes at 15 p.s.i.g.                               ______________________________________                                    

The medium was fed to the 8.5 liter chemostat under the same conditionsas in Example 5 except that the pH was varied between 3.5 and 6.0 andgrowth rate throughout 0.1 hr⁻¹. The following result was obtained:

    ______________________________________                                                                    NPU     NPU                                                                   based   based                                                  TN %  AN %     on TN   on AN                                     ______________________________________                                        Product grown at pH 4.0                                                                      7.8     6.6      54    67                                      Product grown at pH 5.0                                                                      8.6     7.1      57    71                                      Product grown at pH 6.0                                                                      7.7     5.9      61    80                                      ______________________________________                                    

EXAMPLE 6(b)

The culture medium and conditions were as in Example 6 except that thepH was held at 5.0 throughout the run and the temperature was variedbetween 26° and 34° C. The optimum temperature was found to be 30°-32°C.

Examples 7 to 12 describe the fermentation of five variants or isolatesof Fusarium graminearum Schwabe I.M.I. 145425.

EXAMPLE 7

Duplicate shake flasks of 1-liter capacity were prepared containing 200mls. of the following medium:

    ______________________________________                                                                Final                                                                         concentration                                                                 %                                                     ______________________________________                                        Solution 1                                                                            Glucose (sterilised                                                           separately pH 3.0                                                             10 p.s.i./10 min.)    3.0                                             Solution 2                                                                            Ammonium sulphate     0.565                                                   Potassium Dihydrogen  1.0                                                     Phosphate                                                                     MgSO.sub.4 :7H.sub.2 O                                                                              0.025                                                   FeSO.sub.4 :(NH.sub.4).sub.2 SO.sub.4 :6H.sub.2 O                                                   0.0005                                                  MnSO.sub.4 :4H.sub.2 O                                                                              0.0005                                                  CuSO.sub.4 :5H.sub.2 O                                                                              0.0001                                                  CoCl.sub.2 :6H.sub.2 O                                                                              0.0001                                                  CaCl.sub.2 :2H.sub.2 O                                                                              0.0015                                                  Na.sub.2 MoO.sub.4 2H.sub.2 O                                                                       0.00001                                                 NaOH                  0.2                                                     Salts sterilised at 15 p.s.i.g./15 min.                                       Final pH 6.0                                                          Solution 3                                                                            Vitamins as described below were                                              sterilised by filtration                                              ______________________________________                                    

The solutions were added aseptically to give a final volume of 200 ml.then the flasks were inoculated with washed spores of our strain ofFusarium graminearum I-7 I.M.I. 154209 to give a concentration of 8×10³/ml.

The conditions of growth were temperature 30° C., 140 r.p.m. on orbitalshaker with 2" throw.

At hourly intervals the growth was measured by measuring the OpticalDensity of a sample of 600 mμ. From the results obtained the followinggrowth rates were established.

    ______________________________________                                                             Growth rate h.sup.-1                                     ______________________________________                                        (i)   Solution 3 omitted (minimal                                                                        very slow                                                medium)                                                                 (ii)  Solution 3 such that the final                                                                     0.22                                                     concentration of Biotin in                                                    the culture medium was 50 μg/1                                       (iii) Solution 3 such that the final                                                                     0.27                                                     concentration of Biotin in the                                                culture medium was 50 μg/1                                                 and Choline chloride 50 μg/1.                                        ______________________________________                                    

EXAMPLE 8

The procedure of Example 7 was repeated but the strain I-7 was replacedby our strain of Fusarium graminearum Schwabe I-8. The following growthrates were established:

    ______________________________________                                                    Growth rate h.sup.-1                                              ______________________________________                                        (i)      As 7(i)  very slow                                                   (ii)     As 7(ii) 0.22                                                        (iii)    As 7(iii)                                                                              0.27                                                        ______________________________________                                    

EXAMPLE 9

The procedure of Example 7 was repeated but the strain I-7 was replacedby our strain of Fusarium graminearum Schwabe I-9. The following growthrates were established:

    ______________________________________                                                    Growth rate h.sup.-1                                              ______________________________________                                        (i)      As 7(i)  very slow                                                   (ii)     As 7(ii) 0.21                                                        (iii)    As 7(iii)                                                                              0.27                                                        ______________________________________                                    

EXAMPLE 10

The procedure of Example 7 was repeated but the strain I-7 was replacedby our strain of Fusarium graminearum Schwabe I-15. The following growthrates were established:

    ______________________________________                                                    Growth rate h.sup.-1                                              ______________________________________                                        (i)      As 7(i)  very slow                                                   (ii)     As 7(ii) 0.21                                                        (iii)    As 7(iii)                                                                              0.27                                                        ______________________________________                                    

EXAMPLE 11

The procedure of Example 7 was repeated but the strain I-7 was replacedby our strain of Fusarium graminearum Schwabe I-16. The following growthrates were established:

    ______________________________________                                                    Growth rate h.sup.-1                                              ______________________________________                                        (i)      As 7(i)  very slow                                                   (ii)     As 7(ii) 0.21                                                        (iii)    As 7(iii)                                                                              0.27                                                        ______________________________________                                    

EXAMPLE 12

The procedure of Example 7 was repeated but the strain I-7 was replacedby the parent strain Fusarium graminearum Schwabe I.M.I. 145425. Thefollowing growth rates were established:

    ______________________________________                                                    Growth rate h.sup.-1                                              ______________________________________                                        (i)      As 7(i)  very slow                                                   (ii)     As 7(ii) 0.22                                                        (iii)    As 7(iii)                                                                              0.27                                                        ______________________________________                                    

Examples 13 and 14 describe fermentation using strains of Fusaria otherthan Fusarium graminearum.

EXAMPLE 13

A spore suspension of Fusarium solani strain A7-16 (I.M.I. 154217) wasinoculated into a seed fermenter of 80 liter volume containing aglucose, corn steep liquor medium. After growing up the seed fermenterto 10 to 20 gms. per liter, it was split in two, 40 liters to each 400liter vessel. The seed was inoculated into a medium of the followingcomposition:

    ______________________________________                                                        %                                                             ______________________________________                                        Starch            6.0                                                         KH.sub.2 PO.sub.4 0.20                                                        (NH.sub.4).sub.2 SO.sub.4                                                                       0.25                                                        Corn Steep Liquor 0.50                                                        (50% Total Solids)                                                            ______________________________________                                    

pH was 5.5 maintained by addition of sterile ammonia. Temperature 30° C.Pressure 30 p.s.i.g. Air rate 1.0 v.v.m. The revolutions of the stirrerwere increased steadily from 92 to 184 r.p.m. to maintain dissolvedoxygen in the vessel. The agitator consisted of two turbines 0.4D. Whenthe carbohydrate had been utilised the grown mycelium was removed fromthe fermenter, filtered, washed with water, centrifuged, and dried in awarm air band drier at 75° C. The dried product had the followingcomposition:

    ______________________________________                                               Total nitrogen                                                                          9.1%                                                                Ash       8.3%                                                         ______________________________________                                    

When fed to rats this material gave an NPUop of 41 based on TotalNitrogen.

EXAMPLE 14

Fusarium oxysporum strain A9-23 (I.M.I. 154214) was grown exactly as inthe previous example except the starch was replaced by cane molasses ata concentration that produced 6.0% sugars.

The dried product had the following composition:

    ______________________________________                                        Total nitrogen                                                                              9.9%                                                            Ash           10.0%                                                           NFUop         47.0       based on Total                                                                Nitrogen                                             ______________________________________                                    

Methods of analysis for Total Nitrogen (TN) Automatic Kjeldahl digestor(Technicon). A. Ferrari, Ann. N.Y. Sci. 87, 792 (1960).

Amino nitrogen (AN) TNBS (modified). N. A. Pinnegar, Technicon Symposium1965, p. 80.

These novel strains have also been deposited at the American TypeCulture Collection, Rockville, Md., and assigned the respective A.T.C.C.numbers:

    ______________________________________                                        Code No.      A.T.C.C. No.                                                    ______________________________________                                        I 0           20334                                                           I 7           20329                                                           I 8           20330                                                           I 9           20331                                                           I 15          20332                                                           I 16          20333                                                           A9-23         20328                                                           A7-16         20327                                                           ______________________________________                                    

We claim:
 1. Biologically pure fungal culture containing a strain ofFusarium graminearum Schwabe I.M.I. 145425 or a mutant or variantthereof in a culture medium in which this strain is present in a culturemedium containing or being supplied with nutrients or additivesnecessary for the sustenance and multiplication of the strain, themedium having a pH between 3.5 and 7 and the temperature of the mediumbeing maintained at a precise value within the range of between 25° and34° C.
 2. Biologically pure fungal culture containing a strain ofFusarium graminearum Schwabe I.M.I. 145425 or a mutant or variantthereof in a culture medium containing essentially growth-promotingnutrient substances, of which carbon in the form of assimilablecarbohydrate constitutes the limited substrate in proliferation.
 3. Anarticle of manufacture comprising a biologically pure edible nontoxicfungal mycelium of a nontoxic strain of Fusarium possessing a high netprotein utilization value of the order of 65 or above based on α-aminonitrogen.
 4. A biologically pure culture of the microorganism Fusariumgraminearum Schwabe, having the identifying characteristics of IMI145425, said culture being capable of producing proliferation of saidmicroorganism in a recoverable quantity upon fermentation in a culturemedium containing essentially growth-promoting nutrient substances,wherein said proliferated organism comprises an edibleprotein-containing substance possessing a high net protein utilizationvalue of at least 65 based on the α-amino nitrogen.
 5. A biologicallypure culture of the microorganism Fusarium graminearum Schwabe, havingthe identifying characteristics of IMI 154209, said culture beingcapable of producing proliferation of said microorganism in arecoverable quantity upon fermentation in a culture medium containingessentially growth-promoting nutrient substances, wherein saidproliferated organism comprises an edible protein-containing substancepossessing a high net protein utilization value of at least 65 based onthe α-amino nitrogen.
 6. A biologically pure culture of themicroorganism Fusarium graminearum Schwabe, having the identifyingcharacteristics of IMI 154210, said culture being capable of producingproliferation of said microorganism in a recoverable quantity uponfermentation in a culture medium containing essentially growth-promotingnutrient substances, wherein said proliferated organism comprises anedible protein-containing substance possessing a high net proteinutilization value of at least 65 based on the α-amino nitrogen.
 7. Abiologically pure culture of the microorganism Fusarium graminearumSchwabe, having the identifying characteristics of IMI154211, saidculture being capable of producing proliferation of said microorganismin a recoverable quantity upon fermentation in a culture mediumcontaining essentially growth-promoting nutrient substances, whereinsaid proliferated organism comprising an edible protein-containingsubstance possessing a high net protein utilization value of at least 65based on the α-amino nitrogen.
 8. A biologically pure culture of themicrrorganism Fusarium graminearum Schwabe, having the identifyingcharacteristics of IMI 154212, said culture being capable of producingproliferation of said microorganism in a recoverable quantity uponfermentation in a culture medium containing essentially growth-promotingnutrient substances, wherein said proliferated organism comprises anedible protein-containing substance possessing a high net proteinutilization value of at least 65 based on the α-amino nitrogen.
 9. Abiologically pure culture of the microorganism Fusarium graminearumSchwabe, having the identifying characteristics of IMI 154213, saidculture being capable of producing proliferation of said microorganismin a recoverable quantity upon fermentation in a culture mediumcontaining essentially growth-promoting nutrient substances, whereinsaid proliferated organism comprises an edible protein-containingsubstance possessing a high net protein utilization value of at least 65based on the α-amino nitrogen.